本文關(guān)鍵詞：產(chǎn)纖溶酶微生物的篩選、鑒定及其纖溶酶特性研究　出處：《吉林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 血栓性疾病 纖溶酶 淡紫擬青霉 酶學(xué)特性 植物內(nèi)生菌

【摘要】：近年來,血栓性疾病嚴(yán)重影響著人類的健康生活,且發(fā)病率和死亡率逐年增高,導(dǎo)致患者對溶栓藥物的需求不斷增加。然而,市售的溶栓藥物種類較少、特異性差、作用時間短、溶栓能力弱、具有副作用,且價格昂貴。因此,尋找或研制高效、低毒、藥效快、特異性高及價廉的溶栓化合物將會對人類健康產(chǎn)生重要意義。本研究對從植物體內(nèi)分離出的高產(chǎn)纖溶酶的菌株淡紫擬青霉(Paecilomyces lilacinus)及其代謝產(chǎn)生的纖溶酶進(jìn)行了研究。(1)從山西黃河邊、紹興、長白山等地區(qū)采集的245份植物標(biāo)本分離獲得植物內(nèi)生菌480株。利用脫脂奶粉-瓊脂糖平板法從中初步篩選出64株具有分泌胞外蛋白酶能力的菌株。利用纖維蛋白-瓊脂糖平板法復(fù)篩獲得8株分泌纖溶酶的菌株。其中菌株HS-387產(chǎn)酶活力較高,可達(dá)266.58U/m L(相當(dāng)尿激酶酶活力)。該菌株經(jīng)形態(tài)學(xué)及分子生物學(xué)鑒定為淡紫擬青霉(Paecilomyces lilacinus)。(2)分析發(fā)酵條件對菌株HS-387分泌纖溶酶活性的影響,結(jié)果顯示其最佳發(fā)酵條件為培養(yǎng)液裝液量為100m L(250m L錐形瓶),培養(yǎng)液(滅菌前)p H值為4.0,真菌接種量為3.0×106CFU,放置28℃,175rpm,培養(yǎng)7天。優(yōu)化后產(chǎn)生的酶活力為707.97U/m L(相當(dāng)尿激酶酶活力),最終酶活力增到2.66倍。(3)將硫酸銨分級沉淀、DEAE-弱陰離子交換層析、SP-弱陽離子交換層析、丁基疏水層析等方法相結(jié)合,分離、純化獲得經(jīng)Native-PAGE凝膠電泳驗證純度較高(96%以上)的纖溶酶,純化后酶活力回收率為37.2%,最終該酶的比活力為6461.75U/mg(以尿激酶為標(biāo)準(zhǔn))。并對獲得的純度較高的纖溶酶進(jìn)行QE質(zhì)譜鑒定、分析。(4)該菌株產(chǎn)生的纖溶酶等電點(diǎn)約為6.6,分子量約為45k Da,p H值5.0-9.0時該酶較穩(wěn)定,p H3.0以下容易造成酶的失活。該酶在30℃-50℃時均具有較高酶活力,最適溫度40℃。典型絲氨酸蛋白酶抑制劑PMSF對該酶的抑制率為60%,推測該酶可能為堿性絲氨酸蛋白酶。該酶水解纖維蛋白原的先后順序是Aα鏈Bβ鏈γ鏈,對Aα鏈的降解最迅速;水解纖維蛋白的先后順序是γ-γ鏈α鏈β鏈,對降解γ-γ鏈的作用較快。這類似于血纖維蛋白溶酶的水解模式,該酶不僅降解纖維蛋白,對于纖維蛋白的前體纖維蛋白原也有降解作用。研究表明其體外溶栓可能是通過直接降解纖維蛋白的方式發(fā)揮作用,不具有或僅有較小的激酶活性。綜上所述,本研究從480株植物內(nèi)生菌中分離篩選獲得一株高產(chǎn)纖溶酶的菌株淡紫擬青霉(Paecilomyces lilacinus),目前尚未見有該菌種產(chǎn)生纖溶酶的報道。研究發(fā)現(xiàn)其產(chǎn)生的纖溶酶可能為堿性絲氨酸蛋白酶,對纖維蛋白和纖維蛋白原都具有降解作用,推測該酶在溶解血栓的同時還可以防止血栓的繼續(xù)形成,從而為開發(fā)溶栓藥物提供了一個潛在的新途徑。
[Abstract]:In recent years, thrombotic diseases seriously affect the health of human life, and the incidence and mortality increased year by year, resulting in increased demand for thrombolytic drugs. However, commercially available thrombolytic drugs less species, poor specificity, short action time, thrombolytic ability is weak, has side effects, and the price is expensive. Therefore, find or develop efficient, low toxicity, quick effect, high specificity and cheap thrombolytic compounds will have important significance to human health. The study on high yield strains isolated from purple enzyme in plants of Paecilomyces (Paecilomyces lilacinus) and its metabolite produced by plasmin was studied. (1) from the Shaoxing Shanxi the Yellow River side. 245, Changbai Mountain and other regions of the plant specimens isolated from endophytic bacteria of 480 strains. By agarose plate method from defatted milk powder were screened 64 strains with the secretion of extracellular protease of bacteria Strain. Using fibrin agarose plate method for screening 8 strains. The strain HS-387 secreted plasminogen activator enzyme activity is high, up to 266.58U/m L (the equivalent of urokinase enzyme activity). The strains by morphological and molecular biological identification of p.lilacinus (Paecilomyces lilacinus). (2) analysis of the influence of fermentation conditions of secretion of plasminogen activator activity the strain HS-387, the results showed that the optimum fermentation conditions for cultivating liquid liquid volume was 100m L (250m L, medium (flask) before sterilization) P H 4, fungi inoculation quantity is 3 * 106CFU, placed 28 C, 175rpm, cultured for 7 days. After the optimization of enzyme activity 707.97U/m L (the equivalent of urokinase enzyme activity), the final enzyme activity increased to 2.66 times. (3) the ammonium sulfate precipitation, DEAE- weak anion exchange chromatography, SP- weak cation exchange chromatography, hydrophobic interaction chromatography combination, separation, purification of Native-PAGE by coagulation Gel electrophoresis verification of high purity (more than 96%) of the purified enzyme, enzyme activity recovery was 37.2%, the specific activity of the enzyme is 6461.75U/mg (with urokinase as a standard). And to obtain the high purity of the enzyme was identified, QE mass spectrometry analysis. (4) the Strains Producing Fibrinolytic Enzyme isoelectric point is about 6.6, the molecular weight is about 45k Da, 5.0-9.0 of the enzyme was stable P H P H3.0, the following is likely to cause the inactivation of the enzyme. This enzyme has high enzyme activity at 30 DEG -50 DEG, the optimum temperature is 40 degrees centigrade. The typical serine protease inhibitor PMSF and inhibition of this enzyme was 60%, that the enzyme is alkaline serine protease. The enzyme hydrolyzed fibrinogen alpha chain is the sequence of A B beta gamma chain chain, degradation of A alpha chain most rapidly; in order to hydrolyze fibrin is gamma gamma chain chain alpha beta chain, the degradation of gamma gamma chain function rapidly. This is similar in the blood fibrin dissolving enzyme The enzyme hydrolysis mode, not only the degradation of fibrin, the precursor fibrin raw also degradation. The research showed that the in vitro thrombolysis may play a role in the degradation of fibrin directly, does not have or only small kinase activity. In summary, this study from 480 strains of endophytic bacteria isolated get a strain with high fibrinolytic enzyme strains lilac Paecilomyces (Paecilomyces lilacinus), it has not been reported. The strains of plasmin research showed that the generated plasmin may be alkaline serine protease of fibrin and fibrinogen degradation have speculated that at the same time, the enzyme in dissolving thrombus can be formed to prevent thrombosis thus, it provides a new way for the development of potential thrombolytic drugs.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q936

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 卜旺雨;齊義軍;魏華;張娟;馬瑾;王明;馬遠(yuǎn)方;;凝膠電泳蛋白質(zhì)組學(xué)蛋白樣品純化方法比較[J];解剖學(xué)報;2013年06期

2 單金津;李靖圓;謝和;;高產(chǎn)纖溶酶突變株的篩選及發(fā)酵條件的優(yōu)化[J];貴州農(nóng)業(yè)科學(xué);2013年06期

3 朱宏;;基層醫(yī)療衛(wèi)生機(jī)構(gòu)常用心血管藥物[J];中國臨床醫(yī)生;2012年05期

4 魏嶸;劉震;文鏡;;紫外分光光度法測定納豆激酶體外纖溶活力[J];中國釀造;2012年01期

5 ;單因素實驗設(shè)計基本方法之一——完全隨機(jī)設(shè)計[J];臨床薈萃;2011年22期

6 李秀昌;韓曦英;孫健;;利用DPS數(shù)據(jù)處理系統(tǒng)進(jìn)行均勻試驗設(shè)計與分析[J];中國衛(wèi)生統(tǒng)計;2010年02期

7 逯京華;孫智杰;;納豆激酶的研究現(xiàn)狀與展望[J];生物技術(shù)通報;2009年08期

8 羅瓊;金紅;譚學(xué)瑞;;血栓形成機(jī)制及治療進(jìn)展[J];心血管康復(fù)醫(yī)學(xué)雜志;2008年01期

9 何曉紅,滕利榮,孟慶繁,劉蘭英;治療血栓藥物的研究進(jìn)展[J];中國藥學(xué)雜志;2005年04期

10 李家增;血栓形成機(jī)制[J];臨床內(nèi)科雜志;2004年12期

相關(guān)碩士學(xué)位論文 前4條

1 曾祥龍;鐮刀菌天然色素的分離純化、結(jié)構(gòu)分析和性質(zhì)[D];江南大學(xué);2012年

2 關(guān)晨晨;豆豉纖溶酶的分離純化、酶學(xué)性質(zhì)的研究及微膠囊的制備[D];東北林業(yè)大學(xué);2010年

3 高占爭;微生物產(chǎn)纖溶酶的研究[D];江南大學(xué);2006年

4 何葉喧;中草藥牛膝、茜草纖溶酶的篩選與分離純化[D];河北大學(xué);2006年

，

本文編號：1421975